Toxoplasma gondii is an important opportunistic pathogen, causing an episode of toxoplasmic encephalitis in approximately one of every thirteen individuals succumbing to AIDS. Approximately 3 percent of individuals HIV positive initially present with toxoplasmosis. The parasite possesses a unique organelle called the apicoplast, which is genetically and functionally related to plastids such as chloroplasts. Because the human host lacks a similar organelle, the apicoplast is thought to represent an important potential target for the development of anti-parasitic agents. Little is known, however, about the function and biogenesis of the apicoplast. This proposal outlines experiments aimed at characterizing the mechanisms by which proteins are targeted to the apicoplast. We and others have recently shown that proteins destined for the stroma of the T. gondii plastid initially enter the parasite secretory system and then are routed to the plastid. We have generated three models of trafficking to the apicoplast that will be tested in the proposed studies. We will exploit ligand- regulated aggregation to examine the flow of proteins from the endoplasmic reticulum to the apicoplast. We will also identify an apicoplast membrane marker protein and study it at the biochemical and genetic level. The proposed experiments will provide additional insight into the biogenesis of the apicoplast and its importance to T. gondii and related pathogens.